Projets de recherche

Code: 22C05MT

Higenamine is a non-selective β2-agonist, which has been described as a key component of a great variety of natural plants such as Nandina domestica, Tinospora crispa, and Annona squamosa. Although higenamine has never been approved as a drug by the US Food and Drug Administration (FDA), it plays an important role in traditional Chinese herbal medicine (it has been approved for clinical research by the China Food and Drug Administration - CFDA), and has been found to be an ingredient - often unlabeled - of different weight loss and sports supplements, which has been suspected or proven to have caused cases of unintentional anti-doping rule violations.
Within the scope of a previous elimination study aiming at the urinary quantification of higenamine after administration of tropical fruit plants from the Annonaceae family, it could be shown that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. The aim of this project is to conduct further investigation which complement the previous datasey. For that purpose, stability evaluation of componuds of interest, and specific contribution of the different higenamine conjugates will be performed. Besides, two different administrations studies with a total of 6 healthy volunteers  (3 x ♂, 3 x ♀) are planned. Each participant will eat 3 servings on Annona fruit puree with 24 h (8:00 am, 6:00 pm, and next day 8:00 am) and, at after washout, a single dose of two different higenamine-containing nutritional supplements. Urine samples will be collected up to three days following administration in both study phases, and comprehensive analyses are planned to support result management and decision-making processes in case of higenamine findings in sports drug testing programs, preferably by means of new potential biomarkers of plant-derived higenamine administrations.

Code: 22B06XZ

Upon our previous research, a variant in the EPO gene can encode a variant EPO (VAR-EPO) which has a similar molecule weight with recombinant EPO (rEPO), thus cannot be distinguished using the current method of rEPO analysis. Though WADA has established a detailed procedure for investigation and further analysis, it is a burden for both laboratories, Testing Authoity and WADA, nevertheless, rEPO abused by this variant carriers can be hardly detected. For this situation, we are going to develop an effective confirmation method that can discriminate the wild-type EPO, VAR-EPO and rEPO directly, by removing or eliminating the detection of VAR-EPO using the VAR-specific antibody. Two methods will be developed and validated in this project:: reverse-normal immunopurification and step-recognition western blotting. The rEPO administration study on individuals who carry this varient will also be conducted. The collected positive urine and blood samples will be used to evaluate the method. With out goals, the new method can not only protect the "clean" athlete with that variant but also effectively penalize the rEPO abused athelete whether he or she carries that variant. Another significant advantage, this method would be easily adopted by other WADA accredited anti-doping laboratories.

Code: 22B05PC

Erythropoietin receptor agonists (ERAs), e.g. darbepoetins (dEPO), erythropoietins (EPO), EPO-Fc were listed in WADA's Prohibited List under Class S2.1. Traditional polyacrylamide gel-electrophoretic analytic methods were applied by most of the WADA certified laboratories for ERAs detection for the last 20 years. In this project, a novel photoclick hydrogel electrophoretic and in situ immunoblotting method for the screening of ERAs in urine and blood samples of athletes will be developed with high sensitivity and high throughput. A tetrazole-functionalized photoclick hydrogel based chip will be designed first which can satisfy the processing of 15 samples simultaneously. Optimization of the parameters in SAR-PAGE and intensity immunoblotting assay will be carried out on this chip to realize the separation of ERAs, especially the distinguishment of endogenous EPO and recombinant human EPO )rhEPO). Along with the confocal microscope for image acquisition, the LOD of this method can be lower than 1 IU/L (plasma) and 0.1 IU/L (urine) with limited sample volumes. Meanwhile, the whole analysis time can be reduced within 6 hours for plasma samples. Finally, an intravenous administration of rhEPO drugs on healthy subjects will be carried out to verify the practicability of this new method.

Code: 22A10JP

There is a high demand for rapid screening methods using mass spectrometry (MS) that can decrease the turnaround time, cost, and limits of quantitation of existing methodologies. It is important to emphasize that appropriate sample preparation is required to perform proper sample cleanup and analyte enrichment. We are focused on using matrix-compatible SPME-based devices for direct immersion extraction of small molecules from biofluids and direct coupling to MS. In this context, matrix-compatible SPME-based devices have extraction phases comprised of high-capacity sorbent embedded in a polyacrylonitrile binder that ensures small molecules can be extracted while minimizing the co-extraction of macromolecules. This provides better performance and more sensitive analysis of a wide range of small molecules including many of those found on the 2022 Prohibited List. Futhermore, we are focused on couple SPME-based devices to MS via the microfluidic open interface (MOI) and coated blade spray (CBS) technology, as well as developing more efficient ways to separate isobaric/isomeric analytes in the gas phase before MS detection using ion mobility spectrometry (IMS). These developments are critical to decrease the detection limits and discern between permitted and prohibited substances while increasing the speed of analysis and the range of screened and quantified molecules. In this proposal, we build on this success and propose applying SPME technologies directly to MS which have been recently developed in our laboratory to further improve the speed of screening directly during sports events. In the long term, the goal of the team's research is to take advantage of ongoing advances to develop powerful new analytical technologies to assist in the ongoing fight against doping in sport, with an emphasis on lowering the detection limits to better distinguish between permitted from prohibited use, as well as improving the detection window of prohibited substances.

Code: 22A08XT

For substances produced endogenously and included in the WADA Prohibited List, its abuse is based on: 10 identification of the best biomarker of abuse to be applied at the ITP level and 2) confirmation by a procedure able to demonstrate the application of the drug or its origin. Usually, this is performed by establishing the population distribution of the biomarker in ITP and the application isotope ratio mass spectrometry (IRMS) as a confirmatory procedure. This has been applied successfully using the steroidal module of the ABP and GC-IRMS.
In addition to steroidal hormones, several other prohibited compounds may benefit from a similar approach. We intend to investigate the application of IRMS coupled to liquid chromatography to the confirmation of compounds that because of their physicochemical properties cannot be analyzed by GC and for which a definiticive confirmation procedure does not exist.
The analysis of AICAR by GC-C-IRMS has been described after silylation and its comparison with a steroid (pregnanediol) as an endogenous reference compound (ERC). The current limitations of this approach can be overcome by the use of LC-IRMS, allowing the underivatized compound analysis and the use of an ERC closer to its metabolic path.
The main 2-phenethylamine and octopamine target analytes are excreted as sulfates, whose hydrolysis is not obvious. The confirmation by GC-C-IRMS is impaired by the high interaction with GC systems. The direct analysis of their sulfated conjugates by LC-IRMS will unambiguously confirm their origin. The main objective is to prove the applicability for antidoping purposes of LC-IRMS in the analyses of intact sulfates.
For IGF-1 the main objective is the use of LC-IRMS for the analysis of recombinant preparations and preparations of human origin, to prove its applicability for anti-doping purposes. The protein will be analyzed intact or after trypsin digestion.

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